Project Summary We are proposing to continue our development of polarized light microscopy techniques for analyzing the architectural dynamics inside living cells, tissues and whole organisms. We propose to add comprehensive polarization analysis to two multi-view imaging techniques, the dual-view plane illumination microscope, or diSPIM, and the light-field microscope. Both types of polarized light microscopes represent significant new developments that for the first time enable the measurement of molecular orientation parameters in three dimensions at high spatial (0.5m in all three dimensions) and temporal (faster than 1Hz) resolution. The project includes the development of the hardware, software and algorithms required for capturing multi-view image data and their tomographic reconstruction into 3-dimensional maps of birefringence and polarized fluorescence. The instrument development is guided by several application projects including the remodeling of the extracellular collagen matrix by fibroblast cells and the spatial coordination in the assembly of higher order septin structures inside eukaryotic cells. Information about the technology, including software components and instrument design that are developed under this grant, will be made available for users and developers on an established web portal called OpenPolScope.org.